SUPPORTING INFORMATION FILE
Polyphasic analysis of a Middle Age coprolite microbiota, Belgium
Sandra Appelt1, Fabrice Armougom1, Matthieu Le Bailly2, Catherine Robert1, Michel
Drancourt1*
1
Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, 13005
Marseille, France.
2
Franche-Comté University, CNRS UMR 6249 Chrono-Environment, 25 030 Besançon,
France.
*Corresponding author:
Michel Drancourt, URMITE, UM63, Faculté de Médecine, 27 Bd Jean Moulin, 13385
Marseille cedex 5, France, Phone (33) 491 38 55 17, Fax (33) 491 38 77 72.
Email: Michel.Drancourt@medicine.univ-mrs.fr
1
SUPPLEMENTARY INFORMATION FILE CONTENTS :
SUPPLEMENTARY NOTE
SUPPLEMENTARY FIGURES AND TABLES
Supplementary Tables S1-S6
Supplementary Figures S1-S7
SUPPLEMENTARY REFERENCES
2
Supplementary note
SUPPLEMENTARY MATERIAL SECTION. Specific PCR-amplifications and Sangersequencing, quantitative real time PCR.
Specific PCR-amplifications and Sanger-sequencing
Suicide PCR –amplification [1] was performed to reinforce the high-throughput
pyrosequencing results and perform target-orientated searches for Ascaris sp., amoebae and
viridae. For the molecular detection of Ascaris sp., a 145-bp region of the cytochrome b gene
was amplified [2]. To detect Acanthamoeba spp., we targeted the Acanthamoeba spp. 18S
rRNA gene [3]. In addition, seven primer pairs specific for the RNA-polymerase gene C of
Bordetella sp., 16S rRNA gene of Mycobacterium kumamotonense, Rickettsia sp., Ehrlichia
sp., Brucella sp., and Bartonella sp. were designed. The primers are summarized in
Supplementary Table S6. Additionally an internal in-laboratory control was included, to
check for PCR inhibitors in the DNA extract. PCR was performed in a 50-µL final volume
containing 1x PCR buffer, 2 µL of 25 mM MgCl2, 200 µM of each d’NTP, 1 μL of 10 pM of
each primer, 31.15 µL of ddH2O, 1 unit of HotStar Taq Polymerase (Invitrogen, Villebon sur
Yvette, France) and 57-112 ng of DNA-extract. The steps of the PCR were an initial
incubation at 95°C for 15 min; 36-40 cycles of denaturation for 95°C for 1 min, annealing for
45 sec at the corresponding primer annealing temperatures, and elongation at 72°C for 90 sec
and a final elongation 72°C for 10 min; all of these steps were performed in a Gene Amp PCR
System 2700 ABI Thermocycler (Applied Biosystems, Villbon sur Yvette, France). The PCR
products were analyzed using a 2 % agarose gel (UltraPureTM agarose, Invitrogen, Villbon
sur Yvette, France) and purified using the QIAquick PCR Purification Kit (QIAGEN,
Courtaboeuf, France). For the F6-R6 and F8-R8 primer pairs, the PCR products were cloned
into the pEGM-T-Easy Vector system I (Promega, Carbonnieres, France) and transferred via
heat shock into competent JM109 Escherichia coli cells according to the manufacturer’s
3
instructions. White colonies were screened on LB-Amp-XGal-IPTG agar plates using the
following M13 PCR primers M13F 5’-GTAAAACGACGGCCAG-3’ and M13R 5’CAGGAAACAGCTATGAC-3’. All of the PCR and cloning products were sequenced in a
final volume of 20 of µL (1 x sequencing buffer, 3.2 pM forward or reverse primer, 4 µL of
BigDye Terminator V1.1 mix (Applied Biosystems), 7.4 µL of ddH2O and 4 µL of PCR
product) after purification using Sephadex Gel Filtration in the ABI PR ISM 3130xl genetic
sequencer (Applied Biosystems, Villbon sur Yvette, France). The sequences were assembled
using the ChromasPro software and compared with reference sequences of the GenBank
database using NCBI BLAST searches.
Quantitative real time PCR amplifications
Quantitative real-time PCR (qPCR) assays targeting various genomic regions of twenty-two
pathogens were performed to examine for their presence in the coprolite specimen. All of the
targeted microorganisms and genes, including the primer pairs and probes, are listed in
Supplementary Table S8. Serial ten-fold dilutions (from 10 -1 to 10-3) of the DNA extract
derived from the coprolite specimen were performed to dilute potential molecular detection
inhibitors in the brown DNA supernatant. The primers and probes were used at final
concentrations of 500 nM and 62.5 nM, respectively. For the qPCR mix, we used a
commercial MasterMix of Eurogentec (Eurogentec, Angers, France) according to the
manufacturer’s instructions. The reactions were performed using a C1000TM Thermal cycler
(CFX96TM Real-Time System, BIORAD, Marnes-la-Coquette, France). Negative controls
consisting of the reaction mix without DNA were added in a ratio of 1:3. To avoid
contamination, the experiments were performed in working areas in which these systems had
never been used. Overall, 30 different qPCR diagnostic systems were tested using various tenfold dilutions of the coprolite DNA extract. Altogether, 30 different qPCR diagnostic systems
4
were tested using various ten-fold dilutions of the coprolite DNA extract. All of the tested
systems and all of the negative controls remained negative.
5
Table S1. Typical gastro-intestinal, environmental and pathogenic bacteria assigned to the coprolite metagenome#.
Gastro-intestinal Bacteria
Species
A. salmonicida
A. veronii
Bacteroides (82)
B. cellulosilyticus
B. fragilis
B. intestinalis
B. plebeius
Bacteroides sp.
Clostridium (45)
C. botulinum
C. difficile
C. leptum
Collinsella (5)
C. aerofaciens
Corynebacterium (25)
C. aurimucosum
Eikenella (6)
E. corrodens
E. billigiae
Enterobacter (15)
E. cancerogenus
E. cloacae
Enterobacter sp.
Escherichia (27)
E. albertii
E. coli
E. fergusonii
Eubacterium (6)
E. limosum
Flavobacterium (58)
F. johnsoniae
Lactobacillus (11)
L. jensenii
L. buchneri
L. fermentum
Parabacteroides ( 5)
P. merdae
Prevotella (19)
P. ruminicola
P. buccae
P. denticola
P. disiens
Roseburia (42)
R. intestinalis
Roseburia sp.
Ruminococcus (7)
R. albus
Ruminococcus sp.
Serratia (14)
S. odorifera
S. proteamaculans
Streptococcus (8)
S. agalactiae
Genus (Reads)
Aeromonas (7)
Reads
4
3
6
5
5
5
30
6
3
4
4
10
3
3
1
9
5
3
21
3
4
28
3
2
3
3
1
4
4
4
1
41
2
4
8
6
3
Environmental Bacteria
Species
A. avenae
A. citrulli
A. delafieldii
Agrobacterium (199)
A. radiobacter
A. tumefaciens
A. vitis
Beutenbergia (10)
B. cavernae
Burkholderia (832)
B. cenocepacia
B. gladioli
B. multivorans
B. pseudomallei
B. phytofirmans
B. ubonensis
B. graminis
B. phymatum
B. xenovorans
Chromobacterium (25)
C. violaceum
Erythorbacter (170)
E. litoralis
Erythorbacter sp.
Gemmata (59)
G. obscuriglobus
Gemmatimonas (5,370) G. aurantiaca
Laribacteria (16)
L. hongkongensis
Legionella (36)
L. drancourtii
L. pneumophila
Leptospirillum (10)
L. ferrodiazotrophum
L. rubarum
Parachlamydia (31)
P. acanthamoebae
Ralstonia (212)
R. eutropha
R. pickettii
R. solanacearum
Rhizobium (167)
R. etli
R. leguminosarum
Streptomyces (449)
S. bingchenggensis
S. himastatinicus
S. pristinaespiralis
S. scabiei
S. violaceusniger
Genus (Reads)
Acidovorax (76)
Reads
16
7
28
93
41
38
10
36
26
34
41
34
35
46
59
72
25
63
107
57
5,370
16
7
18
8
7
31
110
19
69
144
123
26
37
19
27
26
Bacterial Pathogens
Species
B. henselae
B. rochalimae
Bartonella sp.
Bordetella (153)
B. avium
B. bronchiseptica
B. parapertussis
B. pertussis
Brucella (164)
B. abortus
B. melitensis
B. neotomae
B. ovis
B. suis
Chlamydia (7)
C. pneumoniae
C. trachomatis
C. psittaci
Coxiella (13)
C. burnetii
Granulibacter (45)
G. bethesdensis
Klebsiella (10)
K. pneumoniae
Leptospira (19)
L. borgpetersenii
Mycobacterium (113)
M. intracellulare
M. tuberculosis
M. kansasii
Salmonella (14)
S. enterica
Vibrio (43)
V. cholerae
V. metschnikovii
Yersinia (26)
Y. pseudotuberculosis
Y. ruckeri
Y. kirkensenii
Genus (Reads)
Bartonella (9)
Reads
1
3
3
15
37
16
14
9
10
2
4
5
1
3
2
13
45
4
6
9
9
9
14
7
5
3
4
7
Opportunistic Pathogens
Genus (Reads)
Species
Achromobacter (114)
A. xylosoxidans
Pathogens
A. piechaudii
Actinobacter (20)
A. baumannii
Aeromonas (9)
A. veronii
A. hydrphila
Bacillus (112)
B. cereus
B. thuringiensis
Burkholderia (832)
B. gladioli
B. mallei
B. pseudomallei
Clostridium (45)
C. botulinum
Comamonas (20)
C. testosteroni
Delftia (23)
D. acidovorans
Escherichia (27)
E. coli
Legionella (36)
L. pneumophila
L. drancourtii
Parachlamydia (31)
P. acanthamoebae
Pseudomonas (292)
P. aeruginosa
P. putida
P. stutzeri
Rhodococcus (65)
R. equi
R. erythropolis
Stenotrophomonas
S. maltophilia
(92)
Micrococcus (16)
M. luteus
#
Metagenomic reads were blasted against the NCBI protein database using a translated nucleotide query. Underrepresented taxonomic genera are not
shown.
6
Reads
80
34
4
3
2
14
7
26
4
41
6
19
22
21
18
7
31
47
29
37
21
8
53
16
Table S2. Number and size of contigs that were assigned to (A) Bacteroides spp. and (B) to bacterial pathogens associated to the coprolite sample#.
contig00127°
Contig
length (bp)
555
contig00570*
195
ligand-gated channel protein [Bacteroides coprosuis]
5 E-05
WP_006744840.1
45%
contig01043*
198
GTPase HflX [Bacteroides coprosuis]
1E-08
WP_006744031.1
72%
contig02059°
291
collagen-binding protein [Bacteroides vulgatus]
1E-17
WP_005850005.1
65%
contig01426°
576
bacterial group 2 Ig-like protein [Bacteroides coprocola CAG:162]
9E-17
CDA71009.1
36%
E-value
Hit accession ID
hypothetical protein BPP0674 [Bordetella parapertussis 12822]
1e-32
NP_883015.1
Percent
ID
52%
A)Conti ID
B)Conti ID
E-value
Hit accession ID
TonB-dependent receptor [Bacteroides finegoldii]
3E-13
WP_017142870.1
Percent
ID
37%
Hit description
contig03375
Contig
length (bp)
504
Hit description
contig03491
933
hydrolase [Bordetella bronchiseptica 253]
2e-72
YP_006966397.1
53%
contig02527
503
methyltransferase [Coxiella burnetii RSA 493]
2e-65
NP_819712.1
68%
contig01914
1,568
Possible toxin VapC46. Contains PIN domain [Mycobacterium tuberculosis H37Rv]
8e-08
NP_217901.1
42%
contig03497
497
putative transmembrane protein [Mycobacterium abscessus]
2e-10
WP_005102565.1
30%
contig00341
529
hypothetical protein [Brucella abortus]
3e-62
WP_006089712.1
80%
contig00638
652
Initiation factor 3 [Brucella abortus S19] and
1e-24
YP_001935939.1
79%
contig01163
1,103
hypothetical protein bgla_1g34580 [Burkholderia gladioli BSR3]
5e-37
YP_004362017.1
42%
contig01000
1,019
hypothetical protein BTI_2181 [Burkholderia thailandensis MSMB121]
1e-78
YP_007918661.1
70%
contig02486
704
paraquat-inducible protein B [Granulibacter bethesdensis CGDNIH1]
3e-33
YP_744011.1
49%
contig01542
525
cation efflux transporter [Legionella pneumophila subsp. pneumophila]
6e-06
YP_006506440.1
45%
contig01760
833
Fic/DOC family protein [Leptospira borgpetersenii]
4e-75
WP_002734514.1
52%
contig02804
1,059
perosamine synthetase [Clostridium botulinum]
6e-22
WP_003367053.1
70%
contig03148
808
methionyl-tRNA formyltransferase [Clostridium botulinum A3 str. Loch Maree]
1e-34
YP_001788030.1
36%
contig01373
1,031
6-carboxy-5,6,7,8-tetrahydropterin synthase [Vibrio cholerae]
2e-37
WP_000980008.1
75%
contig01064
537
hypothetical protein [Legionella drancourtii]
2e-27
WP_006872680.1
40%
contig00846
1,349
CP4-6 prophage [Yersinia kristensenii]
2e-124
WP_004390432.1
63%
contig01361
976
DNA methylase N-4/N-6 domain protein [Yersinia kristensenii]
3e-140
WP_004390429.1
65%
847
regulatory protein ada [Parachlamydia acanthamoebae UV-7]
5e-68
YP_004652478.1
64%
contig03337
The contig identifier, its length (bp) and the annotation according to the best BLAST hit (BLASTX versus the non-redundant NCBI database, E-value<1e-05) are summarized. The E-
#
value, the hit accession identifier and the percent of identity are also provided. Contigs of °human and *pig gut microbiota Bacteroides species.
7
Culture
condition
Cultured microorganism
Mode of identification
Schaedler/R2A broths
Stenotrophomonas maltophilia
Micrococcus luteus
Paenibacillus macerans
Bacillus jeotgali
Staphylococcus pasteuri
Staphylococcus epidermidis
Staphylococcus cohnii
Pseudomonas geniculata
Bacillus horti
Clostridium magnum
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
16S rRNA gene sequencing
16S rRNA gene sequencing
Present in highthroughput
pyrosequencing
dataset
yes
yes
no
yes
no
no
no
no
no
no
Sheep blood culture
bottles
Table S3. Cultured microorganisms#
Paenibacillus macerans
Paenibacillus thiaminolyticus
Enterobacter cloacae
Staphylococcus arlettae
Propionibacterium acnes
Paenibacillus ehimensis
Paenibacillus sp.
Rhodanobacter sp.
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
MALDI-TOF
16S rRNA gene sequencing
16S rRNA gene sequencing
yes
no
yes
no
no
no
no
no
#
Reported are the culture conditions, the cultured microorganisms and the mode leading to species identification. Reported is also if the cultured bacteria were
found in the high-throughput pyrosequencing dataset. ° When the identification was based on BLAST annotation of the amplified 16S rRNA gene region
additional phylogenetic trees were constructed (Figures S2).
8
Table S4. Bacterial pathogens identified from the amplified 16S rRNA V6 region#.
Genus
Mycobacterium
Bartonella
#The
Species
M. kumamotonense
B. henselae
B. quintana
B. tribocorum
Reads
1
47
53
1
Size (bp)
240
203-251
270-375
306
Reads > 230 bp
1
19
53
1
species level was defined with a minimum sequence identity of 98.7% using BLAST similarity searches against RDP databases.
9
Table S5. Primers used to amplify DNA from intestinal parasites, bacterial pathogens and amoebae.
Desired specificity
Ascaris sp.
Gene
cytb
Acanthamoeba sp.
18S rRNA
Bordetella sp.
rpoC
Bordetella sp.
rpoC
Bartonella sp.; Brucella sp.
rRNA_BAA
Bartonella sp.; Brucella sp.;
rRNA_rrsC
Bartonella sp.; Brucella sp.
Rickettsia sp.; Erlichia sp.
BQ
Bartonella henselae
Spacer
Spacer
Spacer
Coxiella burnetii
spacer
spacer
Legionella sp.
rpoB
Mycobacterium sp.
IS6110
rpoB
M. kumamotonense
16s rRNA
M. tuberculosis
MTC4
Name
Asc1
Asc2
IDP1
IDP2
F5
R5
F6
R6
F7
R7
F8
R8
F12
R12
Sequence 5'→3'
GTTAGGTTACCGTCTAGTAAGG
CACTCAAAAAGGCCAAAGCACC
TCTCACAAGCTGCTAGGGAGTCA
TCTCACAAGCTGCTAGGGAGTCA
ATGGCGAGGAAGTGCGCCAG
CCGTCCGGCTTGGCCATCAG
GGCGAGCTCAAGGCCACCAG
CAGGTCGGAGGTCGCGAAGC
GCTGGCGCCCCTGCTTCAAA
CCCCGCTGTCTCCAACGCAG
TGGCCTGCGATCTATGTTCT
AGTCGTAACAAGGTAGCCGT
ACAAAGGCTAGCGCCCCTGC
TCCCCGTGAAGATGCGGGGT
Tm (C°)
53
Reference
[2]
60
[3]
60
present study
62
present study
60
present study
53
present study
60
present study
S1 F
S1 R
S3 F
S3 R
S9F
S9R
Cox 2F
Cox 2R
Cox 5F
Cox 5R
TTGCAAAGCAGGTGCTCTCC
TAAGCGTGAGGTCGGAGGTT
CAATGGAGGCAACCGTTCTT
GTGATATCGGGTACATTTTCAACTG
CAACTTCACTGATTTCTGCGATAA
CGAGGAGTGGTTAATATGACAGCT
CAACCCTGAATACCCAAGGA
GAAGCTTCTGATAGGCGGGA
CAGGAGCAAGCTTGAATGCG
TGGTATGACAACCCGTCATG
58
diagnostical tool
49
diagnostical tool
47
diagnostical tool
59
diagnostical tool
59
diagnostical tool
RL1
RL2
ISMtubF
ISMtubR
MF
MR
BF3
BR3
MTC4F
GATGATATCGATCAYCTDGG
TTCVGGCGTTTCAATNGGAC
CCTGCGAGCGTAGGCGTCGG
CTCGTCCAGCGCCGCTTCGG
CGACCACTTCGGCAACCG
TCGATCGGGCACATCCGG
GCGGGTTTTCTCGCAG
GCTCTTTACGCCCAGTAATTC
ATGGGTTCGCCAGACGGCGAG
55
diagnostical tool
68
present study
60
diagnostical tool
50
present study
60
diagnostical tool
10
Rickettsia sp.
dksA
mppA
rpmE
Streptococcus sp.
rpoB
Vibrio cholerae
ompW
cholera toxin
chromosom 1
SodB
Yersinia pestis
glpD
MTC4R
dksA-F
dksA-R
mppA-F
mppA-R
rpmE-F
rpmE-R
Strepto_F
Strepto_R
ompWtsense
ompWantisense
ctxA-s
ctxA-a-s
O1-F
O1-R
SodB-F
SodB-R
glpD-F1
glpD-R2
GATCAGCTACGGGTTGGCCG
TCCCATAGGTAATTTAGGTGTTTC
TACTACCGCATATCCAATTAAAAA
GCAATTATCGGTCCGAATG
TTTCATTTATTTGTCTCAAAATTCA
TTCCGGAAATGTAGTAAATCAATC
TCAGGTTATGAGCCTGACGA
AARYTIGGMCCTGAAGAAAT
TGIARTTTRTCATCAAACATGTG
CACCAAGAAGGTGACTTTATTGTG
54
diagnostical tool
54
diagnostical tool
54
diagnostical tool
46
diagnostical tool
48
[4]
50
[5]
50
[5]
50
[6]
58
[7]
GGTTTGTCGAATTAGCTTCACC
TCAGACGGGATTTGTTAGGCACG
TCTATCTCTGTAGCCCCTATTACG
GTTTCACTGAACAGATGGG
GGTCATCTGTAAGTACAA
AAGACCTCAACTGGCGGTA
GAAGTGTTAGTGATCGCCAGAGT
GGCTAGCCGCCTCAACAAAAACAT
GGTGCCAGTTTCAGTAACAC
11
Table S6. The quantitative real-time PCR systems that were tested.
Microorganism
Brucella sp.
GENE
IS711
IS711
Name
Brucellad
Brucellar
Brucellap
SEQUENCES 5'→3'
GCTCGGTTGCCAATATCAATG
GGGTAAAGCGTCGCCAGAAG
6FAM-AAATCTTCCACCTTGCCCTTGCCATCA
Brucella abortus
IS711
IS711
Abortusd
Abortusr
Abortusp
GCGGCTTTTCTATCACGGTATTC
CATGCGCTATGATCTGGTTACG
6FAM-CGCTCATGCTCGCCAGACTTCAATG
Brucella melitensis
IS711
IS711
Melitensisd
Melitensisr
Melitensisp
AACAAGCGGCACCCCTAAAA
CATGCGCTATGATCTGGTTACG
6FAM-CAGGAGTGTTTCGGCTCAGAATAATCC
Bartonella quintana
yopP
1ère intention
B qui 11580F
B qui 11580R
B qui 11580P
TAAACCTCGGGGGAAGCAGA
TTTCGTCCTCAACCCCATCA
6FAM- CGTTGCCGACAAGACGTCCTTGC -TAMRA
fabF3
fabF3
B qui 05300F
B qui 05300R
B qui 05300P
GCTGGCCTTGCTCTTGATGA
GCTACTCTGCGTGCCTTGGA
6FAM- TGCAGCAGGTGGAGGAGAACGTG -TAMRA
Bartonella grahamii
badA2
1ere intention
B_gra_3_F
B_gra_3_R
B_gra_3_P
AGATGGAAAAATCCGCTCCA
AGGCAAGGGCAAAGAGCATA
6FAM- TCCGCAACGAGTTCTGGTGGTCA -TAMRA
Bordetella pertussis
Toxine
Toxine
Toxine
Toxpert2d
Toxpert2r
Toxpert2p
CCTACCAGAGCGAATATCTGGCA
GCGTTACCCTGCGGATGTTTT
6FAM-ACCGGCGCATTCCGCCC
Chlamydia pneumoniae
omp2
omp2
omp2
ChPnMGBd
ChPnMGBr
ChPnMGB
GATTCGTCGCTAGTGCGGA
GTCTAACCTTCTTCGCTGTCA
6FAM-ACAAAGCCAGCACCTGTTCCT -Mgb
Chlamydia trachomatis
unknown
unknown
unknown
Chlam-tracho_1_F
Chlam-tracho_1_R
Chlam-tracho_1_P
AGCTCCCAAAGCAACCAGAR
BTGTCGCTGCGTTGGTTTTA
6FAM-CAACAGCACCACCAGCAGCTGC
Coxiella burnetii
IS1111A
IS1111A
IS 1111 0706 F
IS 1111 0706 R
CAAGAAACGTATCGCTGTGGC
CACAGAGCCACCGTATGAATC
12
IS1111A
IS1111 07-06 P
6FAM- CCGAGTTCGAAACAATGAGGGCTG -TAMRA
Escherichia coli
ompG
ompG
ompG
ECOmpGMGBAluId
ECOmpGMGBAluIr
ECOmpGMGB
GCTGCGCGTGCAAATGCG
CATGGTCATCGCTTCGGTCT
6FAM-CATCAGAAACTGAACACCAC -Mgb
Klebsiella pneumoniae
carbapenemase (KPC)
β-lactamase
β-lactamase
KPCall-2F
KPCall-2R
KPCprobeall-2
CGCCGTGCAATACAGTGATA
GCAGAGCCCAGTGTCAGCTT
6FAM- CTCTATCGGCGATACCACGT
New Delhi metallobeta-lactamase-1 (NDM-1)
metallo-β-lactamase
metallo-β-lactamase
NDM1-F
NDM1-R
NDM1
GCGCAACACAGCCTGACTTT
CAGCCACCAAAAGCGATGTC
6FAM- CAACCGCGCCCAACTTTGGC
Legionella pneumophila
gyrB
Lpneumo_gyrB_MB
F
Lpneumo_gyrB_MB
R
Lpneumo_gyrB_MB
P
TGGAACCGGTTTGCATCATA
16S
16S
Lep16SMGBd
Lep16SMGBr
Lep16SMGB
GCGGCGAACGGGTGAGTAA
GGAAAGTTATCCAGACTC
6FAM-ACGTGGGTAATCTT -Mgb
hsp
hsp
Lint_hsp_MBF
Lint_hsp_MBR
Lint_hsp_MBP
TTCTCGCTCCCAGAGTAGACA
TTTTCCAATTGAACTTGAACGTC
FAM- ACTGGCCGACCTTCCAGGTGTAGA
Listeria monocytogenes
hlyQ
hlyQ
hlyQ
hlyQf
hlyQr
hlyQp
CATGGCACCACCAGCATC
ATCCGCGTGTTTCTTTTCGA
6FAM-CGCCTGCAAGTCCTAAGACGCCA
Mycobacterium tuberculosis
ITS
ITS
ITS
ITSd
ITSr
sonde tub
GGGTGGGGTGTGGTGTTTGA
CAAGGCATCCACCATGCGC
6FAM-GCTAGCCGGCAGCGTATCCAT
Rickettsia sp.
unknown
DIAGNOSTIC
DIAGNOSTIC
1029-F1
1029-R1
Rick1029_MBP
GAMAAATGAATTATATACGCCGCAAA
ATTATTKCCAAATATTCGTCCTGTAC
6FAM- CGGCAGGTAAGKATGCTACTCAAGATAA
gltA (CS)
RKND03_F
GTGAATGAAAGATTACACTATTTAT
gyrB
Leptospira interrogans
CGACAGGAATACCACGRCCA
FAM- TGAATCCCTGGCAGGTTATTGCAAGG
13
EPIDEMIO
EPIDEMIO
RKND03_R
RKND03 P
GTATCTTAGCAATCATTCTAATAGC
6FAM- CTATTATGCTTGCGGCTGTCGGTTC -TAMRA
Rickettsia prowazekii
ompB
ompB
ompB
Rpr_ompB_F
Rpr_ompB_R
Rpr_ompB_P
AATGCTCTTGCAGCTGGTTCT
TCGAGTGCTAATATTTTTGAAGCA
6FAM- CGGTGGTGTTAATGCTGCGTTACAACA -TAMRA
Rickettsia felis
bioB
bioB
bioB
R_fel0527_F
R_fel0527_R
R_fel0527_P
ATGTTCGGGCTTCCGGTATG
CCGATTCAGCAGGTTCTTCAA
6FAM- GCTGCGGCGGTATTTTAGGAATGGG -TAMRA
orfb
orfb
orfb
orfb-f
orfb-r
orfb- p (sonde tamra)
CCCTTTTCGTAACGCTTTGCT
GGGCTAAACCAGGGAAACCT
6-FAM-TGTTCCGGTTTTAACGGCAGATACCCA-TAMRA
Shiga toxine de type 2
stx2
stx2
stx2
stx2F
stx2R
stx2P
GTGGCATTAATACTGAATTGTCATCA
GCGTAATCCCACGGACTCTTC
6FAM- CGGACCTCTGTATCTGCCTGAAGCGTAAGGGTCCG
Streptococcus pneumoniae
systeme poc
systeme poc
systeme poc
plyNd
plyNR
plyN_P
GCGATAGCTTTCTCCAAGTGG
TTAGCCAACAAATCGTTTACCG
6FAM-CCCAGCAATTCAAGTGTTCGCCGA
lytA
lytA
lytA
Pneumo_lytA_F
Pneumo_lytA_R
LytA_P
CCTGTAGCCATTTCGCCTGA
GACCGCTGGAGGAAGCACA
6FAM-AGACGGCAACTGGTACTGGTTCGACAA
14
Figure S1. Working overview of the polyphasic approach used to analyze the Namur coprolite.
15
Figure S2. Phylogenetic of 16S rRNA gene sequences generated form cultivated bacteria species. The tree was constructed using the PhyML algorithm
with a bootstrap of 100. The bootstrap support is reported for each branch. Phylogenetic tree of 16S rDNA amplicons closely related to (A) Bacillus
horti, (B) Paenibacillus spp., (C) Rhodanobacter spp. and (D) Clostridium magnum.
16
Figure S3. Phylogenetic tree of a hydrolase.
A phylogenetic tree was generated from the translated open reading frame of a contig encoding a hydrolase close to Bordetella species. The tree was
constructed using the PhyML algorithm with a bootstrap of 100. The bootstrap support is reported for each branch.
17
Figure S4. Bayesian source-tracking results.
(A) The mixture of taxa associated to the 16S rDNA gene amplicon dataset of the coprolite specimen was compared to known dataset of various
environments. To control the workflow used to perform the analyses, two known samples (B) one coprolite previously investigated [9] and (C) a
soil sample [10] were positively tested.
18
Figure S5. Phylogenetic tree of 16S rDNA amplicons matching to Bartonella sp..
The tree was constructed using the PhyML algorithm with a bootstrap of 100. The bootstraps
are reported for each branch. Phylogenetic tree of 16S rDNA amplicons closely related to (A)
B. henselae, B. koehlerae and (B) B. quintana.
19
Figure S6. Alignment and of the amplicon matching to Bordetella and Achromobacter.
The sequence alignment was performed using CLUSTALW multiple alignment tool [11].
20
Figure S7. Metabolic comparison of modern metagenomes to the coprolite metagenome. The Principal coordinates analysis was based on read classification
according to BLASTX searches against the SEED Database. For each metagenome included the MG-RAST accession number is given. Compared metagenomes
are from soil (yellow cluster), healthy mammalian and human feces (blue cluster); and the coprolite (red). The coprolite metagenome does not group with either
the modern gut or soil microbiota.
21
SUPPLEMENTARY REFERENCES
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extraction amplification and sequences from eggs collected in coprolites. Int J Parasitol
31: 1101-1106.
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ribosomal DNA PCR and sequencing for genus and genotype identification of
acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 39:
1903-1911.
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species-specific identification of Vibrio cholerae using primers targeted to the gene of
outer membrane protein OmpW. J Clin Microbiol 38: 4145-4151.
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Vibrio cholerae and ctxA in Peruvian coastal water and plankton by PCR. Appl Environ
Microbiol 69: 3676-3680.
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isolates by a multiplex PCR assay and rpoB sequence determination. J Clin Microbiol 45:
134-140.
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remains of ancient plague patients. Emerg Infect Dis 13: 332-333.
8. Iniguez AM, Reinhard K, Carvalho Goncalves ML, Ferreira LF, Araujo A, et al. (2006) SL1
RNA gene recovery from Enterobius vermicularis ancient DNA in pre-Columbian
human coprolites. Int J Parasitol 36: 1419-1425.
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9. Tito RY, Knights D, Metcalf J, Obregon-Tito AJ, Cleeland L, et al. (2012) Insights from
characterizing extinct human gut microbiomes. PLoS One 7: e51146.
10. Lauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-based assessment of soil
pH as a predictor of soil bacterial community structure at the continental scale. Appl
Environ Microbiol 75: 5111-5120.
11. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific
gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680.
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